Summary Aberrant activation of tyrosine kinases causes dysregulated cell signaling resulting in cancer cell proliferation, invasion, and metastasis. Targeted tyrosine kinase inhibitors are achieving the remarkable success in the field of hematologic malignancies, exemplifying the significance of studying activated tyrosine kinase pathways in human diseases. To understand what pathways are active and may thus be targeted therapeutically, comprehensive analysis of tyrosine phosphorylation of cellular proteins is needed. Currently, tyrosine phosphoproteomics (pTyr-omics) approaches including mass spectrometry and microarray systems are predominantly used as research tools rather than for clinical diagnosis due to technical complexities. The lack of robust and convenient pTyr-omics tools hampers the discovery of biomarkers relevant to tyrosine kinase pathways and the development of new therapeutic strategies. By contrast, flow cytometry has been integrated in clinical laboratories as a routine tool for immunophenotyping of blood cells at the single cell level. In an effort to develop a molecular diagnostic tool based on the global tyrosine phosphorylation state, we developed SH2 profiling in which SH2 domains, a major pTyr binding module in mammalian cells, are utilized as probes to detect changes in the global tyrosine phosphorylation state of cancer cells. Recently, we found that a group of chronic lymphocytic leukemia (CLL) cases selected by SH2 profiling at diagnosis showed high likelihood of disease progression during ~5 years of clinical follow-up. This result suggests the potential to use the SH2 profile as a predictive biomarker, motivating us to develop a new assay platform by which the SH2 profiling can be routinely performed in clinical laboratories with single-cell resolution. Here we propose to develop BCR pathway-focused SH2 profiling assays integrating two new flow cytometry technologies (SH2-flow). In Aim 1, we will prepare a universal B Cell Receptor (BCR) probe library (BCR-SH2 panel) in which fluorescent dyes are covalently and stoichiometrically coupled to SH2 domains, allowing for multiplexed binding assays. In Aim 2, we will develop a multi-color flow cytometry assay of human B lymphocytes using the set of fluorescent SH2 probes. In Aim 3, we will develop an SH2 binding assay based on CyTOF mass cytometer for highly multiplex SH2 profiling at a single cell resolution. Bringing a pathway- focused phosphotyrosine profiling tool to routine clinical practice by complementing existing antibody-based immunophenotyping would have a remarkable impact on clinical research and possibly on patient care. The new assays could be utilized for discovery of signaling-based predictive biomarkers, indicators for disease progression, and tools to assess targeted drug responses.